T. Hoang et al., THE STRUCTURE AND DYNAMICS OF THE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR DEFINED BY THE TERNARY COMPLEX MODEL, The Journal of biological chemistry, 268(16), 1993, pp. 11881-11887
Myeloid cell lines and primary leukemic myeloblasts express two classe
s of granulocyte macrophage colony-stimulating factor (GM-CSF) binding
sites of high (K(d) 20-50 pM) and low affinity (K(d) 5-10 nM). High a
ffinity binding is caused by the association of two chains, p80alpha a
nd p130beta, whereas p80alpha alone confers low affinity binding only.
Furthermore interleukin-3 (IL-3) competes for the binding of GM-CSF t
o its high affinity receptor (for review see Nicola, N. A., and Metcal
f, D. (1991) Cell 67, 1-4). In the present study, we took advantage of
the perturbation of GM-CSF binding equilibrium by IL-3 to take a quan
titative approach to analysis of the structure and dynamics of the GM-
CSF receptor complex. First, cross-linking studies were performed at t
wo concentrations of radioligand. At 200 pM, a concentration sufficien
t for near saturation of the high affinity binding site R1, the associ
ation between p80alpha and p130beta is stoichiometric, and the additio
n of IL-3 prevents the binding to both chains. At 5 nM, a concentratio
n sufficient for half-occupancy of the low affinity binding site R2, I
L-3 prevents cross-linking to the beta chain only. Second, GM-CSF satu
ration curves were analyzed both at equilibrium and under conditions o
f perturbation of the equilibrium by IL-3. In the presence of IL-3, th
e interaction of GM-CSF with its receptor is converted from high to lo
w affinity binding. Computer modeling of binding data with a ternary c
omplex model involving GM-CSF, p80alpha, and p130beta indicates that t
he model fits the data with accuracy and suggests that ligand binding
stabilizes the interaction between p80alpha and p130beta by 3 orders o
f magnitude. Third, membrane solubilization dissociates p80alpha and p
130beta whereas on ligand-stabilized preformed complexes, solubilizati
on did not dissociate the two chains. Finally, upon addition of GM-CSF
, there is an increase with time in the proportion of ligand bound to
the high affinity receptor, at the expense of that bound to low affini
ty receptor, suggesting that stabilization of the ternary complex is a
time-dependent process.